Paracrine factors of vascular endothelial cells facilitate cardiomyocyte differentiation of mouse embryonic stem cells.

For myocardial regeneration therapy, the low differentiation capability of functional cardiomyocytes sufficient to replace the damaged myocardial tissue is one of the major difficulties. Using Nkx2.5-GFP knock-in ES cells, we show a new efficient method to obtain cardiomyocytes from embryonic stem (ES) cells. The proportion of GFP-positive cells was significantly increased when ES cells were cultured with a conditioned medium from aortic endothelial cells (ECs), accompanied by upregulation of cardiac-specific genes as well as other mesodermal genes. The promotion was more prominent when EC-conditioned medium was added at an early stage of ES cell differentiation culture (Day 0-3). Inhibitors of bone morphogenic protein (BMP), cyclooxygenase (COX), and nitric oxide synthetase (NO) prevented the promotion of cardiomyogenesis by EC-conditioned medium. These results suggest that supplementation of EC-conditioned medium enables cardiomyocytes to be obtained efficiently through promotion of mesoderm induction, which is regulated by BMP, COX, and NOS.